Proteins and polypeptides have become increasingly important therapeutic agents. In most cases, these proteins and polypeptides are produced in cell culture, from cells that have been engineered and/or selected to produce unusually high levels of the particular protein or polypeptide of interest. Control and optimization of cell culture conditions is critically important for successful commercial production of proteins and polypeptides.
Many proteins and polypeptides produced in cell culture are glycoproteins that contain covalently linked carbohydrate structures including oligosaccharide chains. These oligosaccharide chains are linked to the protein in the endoplasmic reticulum and the Golgi apparatus via either N-linkages or O-linkages. The oligosaccharide chains may comprise a significant portion of the mass of the glycoprotein. The oligosaccharide chains are thought to play key roles in the function of the glycoprotein including facilitating correct folding of the glycoprotein, mediating protein-protein interactions, conferring stability, conferring advantageous pharmacodynamic and/or pharmacokinetic properties, inhibiting proteolytic digestion, targeting the glycoprotein to the proper secretory pathway and targeting the glycoprotein to a particular organ or organs.
Generally, N-linked oligosaccharide chains are added to the nascent, translocating protein in the lumen of the endoplasmic reticulum (see Molecular Biology of the Cell, by Alberts et al., 1994, incorporated herein by reference). The oligosaccharide is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. The initial oligosaccharide chain is usually trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues.
After initial processing in the endoplasmic reticulum, the glycoprotein is shuttled via small vesicles to the Golgi apparatus, where the oligosaccharide chain undergoes further processing before being secreted to the cell surface. The trimmed N-linked oligosaccharide chain may be modified by the addition of several mannose residues, resulting in a high-mannose oligosaccharide. Alternatively, one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form complex oligosaccharides. Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue. Additionally, a fucose residue may be added to an N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.
In addition to being modified by the N-linked glycosylation pathway, glycoproteins may also be modified by the addition of O-linked oligosaccharide chains to specific serine or threonine residues as they are processed in the Golgi apparatus. The residues of an O-linked oligosaccharide are added one at a time and the addition of each residue is catalyzed by a specific enzyme. In contrast to N-linked glycosylation, the consensus amino acid sequence for O-linked glycosylation is less well defined.
The ultimate quality and extent of protein glycosylation can be dramatically affected by the conditions of the cell culture. For example, traditional batch and fed-batch culture processes have focused on the ultimate level of the peptide produced and often result in production of a glycoprotein with a less extensive glycosylation pattern and/or a glycosylation pattern whose sugar residues of the oligosaccharide chains poorly reflect the sugar residues that are present in the naturally occurring form of the glycoprotein. Increasing the extent of glycosylation and/or adjusting the composition of the sugar residues to more closely reflect the level and composition of glycosylation that are present in the natural form of the glycoprotein could potentially result in a therapeutic glycoprotein agent with greater potency, improved pharmacodynamic and/or pharmacokinetic properties and fewer side effects. While some effort has been made to improve the quality and quantity of glycosylation of glycoproteins produced in cell culture, there remains a need for additional improvements. There is a particular need for the development of systems for producing glycoproteins with improved glycosylation patterns by cell culture in defined media.